Microbial-derived chondroitin sulfate

ABSTRACT

Described is chondroitin sulfate obtained from microbial sources, and related compositions and methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Application No. 61/129,084, filed Jun. 4, 2008, theentire contents of which are incorporated herein by reference in theirentirety.

FIELD OF INVENTION

Described herein is chondrotin sulfate obtained by microbialfermentation, such as from Bacillus natto, and related compositions andmethods.

BACKGROUND

Osteoarthritis (OA) is the most common musculoskeletal disease and itaffects about 10% of the world's population aged 60 and older.Buckwalter, J. A. and Martin, J. A. Adv. Drug Deliv. Rev., 58, 150(2006). OA affects the entire joint and is characterized by a loss ofcartilage. Conventional OA treatment consists of nonsteroidalanti-inflammatory drugs (NSAIDs) and analgesics. Reviewed in, e.g.,Reginster, J-Y., et al. Mini-Reviews in Medicinal Chemistry, 7,1051-1061 (2007). However, many of these drugs can cause serious sideeffects and therefore strategies have been developed for usingchondroitin sulfate as a means for treating OA. Chondroitin sulfate (CS)is widely used to treat OA. See, e.g., Reginster et al., “Symptom andStructure Modifying Properties of Chondroitin Sulfate inOsteoarthritis,” Mini-Reviews in Med. Chem., 7: 1051-61 (2007). CS isclassified as a symptomatic slow acting drug in osteoarthritis(SYSADOAs), acting after a few weeks time, as compared to analgesics andNSAIDs, which act within a few hours.

Chondroitin sulfate (CS) is a major component of the extracellularmatrix, and is found in animal cartilage. CS is a sulfatedglycosaminoglycan (GAG) consisting of repeating units of alternatingglucuronic acid and N-acetyl-galactosamine that contains sulfate groupsat one or more positions on the N-acetyl-galactosamine units, such asthe 4 position (CS A), 6 position (CS C), 2 position (CS D) or the 4 and6 positions (CS E). While the size of CS varies, typical CS moleculeshave a size of about 20,000 to 50,000 daltons. Current sources of CSinclude cartilage from various animals, such as pigs (ear and nose),cows (trachea), sharks, fish and birds.

SUMMARY OF THE INVENTION

In accordance with some embodiments, there is provided methods forproducing a chondroitin sulfate-like compound, comprising (a) culturinga microbial culture under conditions suitable for chondroitin sulfateproduction; and (b) obtaining chondroitin sulfate from the culture. Insome embodiments, the microbial culture comprises a bacterium or afungus, such as a naturally occurring bacterium or a naturally occurringfungus.

In some embodiments, the bacterium is from a genus selected from thegroup consisting of Corynebacterium, Microbacterium, Micrococcus,Monascus, Streptomycetes, Escherichia, Bacillus and Lactobacillus,including Bacillus natto, such as a strain of Bacillus natto selectedfrom the group consisting of the Naruse strain, the Miura strain and theTakahashi strain. In some embodiments, the bacterial culture comprisesnatto.

In some embodiments, the fungus is from a genus selected from the groupconsisting of Aspergillus, Endomycopsella, Endomycopsis, Hansenula,Hasegawaea, Penicillium, Pichia, Monascus, Candida, Debaryomyces,Eurotium, Galactomycetes, Geotrichum, Rhodotorula, Saccharomyces,Trichoderma, Kluveromyces, Schizosaccharomyces, Streptomyces,Talaromyces. Torulopsis, Yamadazyma, Yarrowia, Zygosaccharomyces, Mucor,Mortierella, Rhizomucor, Rhizopus, Cryptococcus, Dipodascus, andTrichosporon.

In some embodiments, the culturing comprises incubating the microbialculture in nutrient broth at 37° C. In some embodiments, the culturingcomprises incubating the microbial culture in culture medium at 30° C.In some embodiments, the culturing comprises two stages: a first stagecomprising culturing in a pre-culture medium and a second stagecomprising culturing in a main culture medium.

In some embodiments, the obtaining comprises a method selected from thegroup consisting of centrifugation to remove microbial cells,filtration, chromatography, and alcohol precipitation.

In accordance with other embodiments, there is provided an isolatedchondroitin sulfate-like compound produced by the methods describedhereinabove and below. In some embodiments, the isolated chondroitinsulfate-like compound is chondroitin sulfate. In some embodiments, theisolated chondroitin sulfate-like compound comprisesN-acetyl-galactosamine groups having a sulfate group at the 4 positionand N-acetyl-galactosamine groups having a sulfate group at the 6position.

In accordance with other embodiments, there is provided a compositioncomprising an isolated chondroitin sulfate-like compound produced by themethods described herein above and below. In some embodiments, thecomposition is a pharmaceutical composition, nutraceutical composition,or food product.

In accordance with other embodiments, there is provided an isolatedmicrobial-derived chondroitin sulfate-like compound. In someembodiments, the chondroitin sulfate-like compound comprisesN-acetyl-galactosamine groups having a sulfate group at the 4 positionand N-acetyl-galactosamine groups having a sulfate group at the 6position, wherein the ratio of disaccharides with sulfate at the 4position (4S) to disaccharides with sulfate at the 6 position (6S) isgreater than 1, or is less than 1. In some embodiments, the chondroitinsulfate-like compound has a molecular weight selected from (i) about 300to about 3,000 daltons; (ii) about 1,000 to about 10,000 daltons; (iii)about 500 to about 15,000 daltons; and (iv) about 1,000 to about 20,000daltons; (iv) about 1,000 to about 25,000 daltons; (iv) about 5,000 toabout 35,000 daltons. In some embodiments, the microbial-derivedchondroitin sulfate-like compound is chondroitin sulfate.

In accordance with other embodiments, there is provided a method for thetreatment or prevention of osteoarthritis, or for the maintenance ofmusculoskeletal health, comprising administering to a patient in needthereof a therapeutically effective amount of a chondroitin sulfate-likecompound produced by the methods described herein above and below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an analysis of the size distribution ofporcine CS by size exclusion chromatography, plotting CS content ascompared to fraction number.

FIG. 2 shows the results of an analysis of the size distribution ofbacteria-derived CS by size exclusion chromatography, plotting CScontent as compared to fraction number.

FIG. 3 shows typical HPLC analysis of CS from bovine trachea (A), sharkcartilage (B), porcine trachea (C), and Bacillus natto Naruse strain(D).

FIG. 4 shows the microbial strains selected from a broth libraryscreening.

FIG. 5 shows ELISA analysis of CS digestion products from bovine,Bacillus natto Naruse strain and porcine CS.

DETAILED DESCRIPTION

The present invention relates to the discovery that CS, or a CS-likecompound, can be obtained from microbial sources, including bacterialsources, such as Bacillus natto, and fungal sources. This is animportant discovery because current methods of obtaining CS from animalsources may not be able to satisfy the increasing demand for CS. Forexample, it would take millions of animals to obtain enough CS tosatisfy a large commercial market, such as Japan. Additionally, CS frommicrobial sources avoids the potential of contamination with animalviruses and prions that may be associated with CS from current animalsources.

Thus, in accordance with some aspects, the present invention providesmicrobial-derived CS or CS-like compound that can be used instead ofcurrently available animal-derived CS. Compositions comprisingmicrobial-derived CS or CS-like compound, and methods of making andusing it, also are provided. The microbial-derived CS or CS-likecompound described herein are free of animal contaminants, includingmammalian contaminants, such as mammalian proteins, viruses and prions,that may be present in CS obtained from cows or pigs.

All technical terms used herein are terms commonly used in biochemistry,molecular biology and agriculture, and will readily be understood by oneof ordinary skill in the art to which this invention belongs.

As used herein, the singular forms “a,” “an,” and “the” designate boththe singular and the plural, unless expressly stated to designate thesingular only.

As used herein, “chondroitin sulfate” denotes a sulfatedglycosaminoglycan (GAG) consisting of repeating units of alternatingglucuronic acid and N-acetyl-galactosamine that contains sulfate groupsat one or more positions on the N-acetyl-galactosamine units.

As used herein, “chondroitin sulfate-like compound” denotes a compoundthat exhibits chondroitin sulfate activity in the dimethylene blue assay(DMMBA) described herein and/or can be digested by chondroitinase anddetected by antibodies to the 4S and 6S dissacharides of CS, asillustrated in FIG. 5. In some embodiments, the chondroitin sulfate-likecompound is chondroitin sulfate. In some embodiments, the

CS-like compound includes N-acetyl-galactosamine groups that containsulfate groups at one or more positions, such as at the 4, 6, 2 or the 4and 6 positions. In some embodiments, the CS-like compound additionallyor alternatively includes glucuronic acid groups. In some embodiments,the CS-like compound includes repeating units of alternating glucuronicacid and N-acetyl-galactosamine groups that contains sulfate groups atone or more positions, such as at the 4, 6, 2 or the 4 and 6 positions.

In some embodiments, the CS-like compound has a size consistent withanimal-derived chondroitin sulfate. In other embodiments the CS-likecompound has a size different from animal-derived chondroitin sulfate.In some embodiments, the CS-like compound has a 4S:6S ratio similar tothat of an animal-derived chondroitin sulfate. In other embodiments, theCS-like compound has a 4S:6S ratio different from that of ananimal-derived chondroitin sulfate.

While animal-derived CS compounds typically have a size between 20,000and 50,000 daltons, microbial-derived CS-like compounds typicallyexhibit a size below 20,000 daltons, as illustrated in Table 2 below,although some microbial-derived CS-like compounds exhibit a size above20,000 daltons, as also illustrated in Table 2. Thus, microbial-derivedCS-like compounds may have a size between 300 and 50,000 daltons, orgreater. In one embodiment, a microbial-derived CS-like compound has amolecular weight between 300 and 3,000 daltons. In another embodiment, amicrobial-derived CS-like compound has a molecular weight between 1,000and 10,000 daltons. In an other embodiment, a microbial-derived CS-likecompound has a molecular weight between 500 and 15,000 daltons. Inanother embodiment, a microbial-derived CS-like compound has a molecularweight between 1,000 and 20,000 daltons. In another embodiment, amicrobial-derived CS-like compound has a molecular weight between 1,000and 25,000 daltons. In another embodiment, a microbial-derived CS-likecompound has a molecular weight between 5,000 and 35,000 daltons. Inanother embodiment, a microbial-derived CS-like compound has a molecularweight up to 50,000 daltons, or greater.

The ratio of N-acetyl-galactosamine groups with sulfates at the 4position (4S) to groups with sulfates at the 6 position (6S) (forexample, as determined by analyzing the disaccharides resulting fromchondroitinase digestion) varies with the source of the chondroitinsulfate. For example, shark chondroitin sulfate has more 6S than 4S,while bovine and porcine chondroitin sulfate have more 4S than 6S.Microbial-derived CS-like compounds exhibit a broad range of 4S:6Sratios, as illustrated in Table 2 below.

This variation may be due in part to the particular microbial productionstrain and/or to the culture conditions. In one embodiment, amicrobial-derived CS-like compound has a 4S:6S ratio of less than 1. Inanother embodiment, a microbial-derived CS-like compound has a 4S:6Sratio of greater than 1. In another embodiment, a microbial-derivedCS-like compound has a 4S:6S ratio of between 1 and 6. In anotherembodiment, a microbial-derived CS-like compound has a 4S:6S ratio ofgreater than 6.

Microbial Sources

A variety of microorganisms can be cultured for CS or CS-like compoundproduction. In some embodiments, the microorganisms are naturallyoccurring microorganisms, such as microorganisms that have not beengenetically engineered or recombinantly transformed to produce CS orCS-like compound. Exemplary microorganisms include but are not limitedto bacteria and fungi.

Illustrative bacteria include but are not limited to phylaActinobacteria (e.g. genera Corynebacterium, Microbacterium,Micrococcus, Monascus, and Streptomycetes), Proteobacteria (e.g. generaEscherichia), and Firmicutes (e.g. genera Bacillus and Lactobacillus).Suitable bacteria include those listed in Table 3 and FIG. 4. Suitablebacteria are well-known in the art and publicly available from, e.g., acommercial supplier, food product, or natural source, such as soil.

One microbial source of particular interest is Bacillus natto, which isa sub-species of Bacillus subtilis. As discussed in more detail in theexamples below, to date three different strains of Bacillus subtilisvar. natto have been identified as producers of CS-like compound: theNaruse, Miura and Takahashi strains.

Bacillus natto currently is used in the commercial production of theJapanese food natto and the similar Korean food cheonggukjang. Anysource of Bacillus natto can be used in the methods described herein,such as Bacillus natto from a commercial supplier or natural source,such as soil. Alternatively, the natto food product, which containsBacillus natto, can be used as a source of Bacillus natto.

Other Bacillus strains also have been found to produce CS or CS-likecompound, as illustrated in the examples below.

Illustrative fungi include but are not limited to phyla Deuteromycota(e.g. genera Aspergillus), Ascomycota (e.g. genera Endomycopsis,Hansenula, Hasegawaea, Penicillium, Pichia, Monascus, Candida,Debaryomyces, Eurotium, Galactomycetes, Geotrichum, Saccharomyces,Trichoderma, Kluveromyces, Schizosaccharomyces, Talaromyces. Torulopsis,Yamadazyma, Yarrowia, and Zygosaccharomyces), Zygomycota (e.g. generaMucor, Mortierella, Rhizomucor, and Rhizopus), Basidiomycota (e.g.genera Cryptococcus, Dipodascus, and Trichosporon). Suitable fungiinclude those listed in Table 4 and FIG. 4. Fungi are well-known in theart and publicly available from, e.g., a commercial supplier, foodproduct, or natural source, such as soil.

Microbial strains that produce CS or CS-like compounds can be identifiedby following the methods outlined in the examples below.

Production Methods

Microbial-derived CS or CS-like compounds can be obtained by culturingany microorganism that produces CS or CS-like compound, includingmicroorganisms that have not been genetically engineered orrecombinantly transformed to produce CS or CS-like compound, such as anaturally occurring CS-producing strain of Bacillus natto.

The microorganism can be cultured under standard conditions. Forexample, bacterial cultures of Bacillus natto may be grown in a nutrientbroth at 37° C. overnight and then the overnight culture may becentrifuged to separate bacterial cells from CS or CS-like compound inthe broth. Alternatively, a sample of natto food product, such as asample of a commercial preparation of natto, may be cultured under thesame or similar conditions to obtain CS or CS-like compound. Fungalmicroorganisms likewise can be cultured under standard conditionsincluding tank or liquid fermentation or solid state fermentation, suchas fermentation on solid wheat bran.

Suitable culture media can be selected by the skilled practitioner basedon the microorganism being cultured. Any type of medium can be used,provided that a CS-producing microorganism can be grown therein. Forexample, a medium to be used may contain carbon sources such as glucose,sucrose, gentiobiose, soluble starch, glycerol, dextrin, molasses andorganic acids; nitrogen sources such as ammonium sulfate, ammoniumcarbonate, ammonium phosphate, ammonium acetate, peptone, yeast extract,corn steep liquor, casein hydrolysate, wheat bran and meat extract; andinorganic salts such as potassium salts, magnesium salts, sodium salts,phosphates, manganese salts, iron salts and zinc salts.

In some embodiments, an inducer can be added to the medium in order toenhance production of the CS-like compound. Examples of suitableinducers include saccharides, such as gentose (e.g., Gentose #80, NihonShokuhin Kako), gentiobiose and gentio-oligosaccharide (e.g.,Gentio-oligosaccharide, Wako Pure Chemicals). The skilled practitionercan determine a suitable amount of inducer to be added depending on thestrain and/or culture conditions. Exemplary amounts include from 0.01 to5%.

In some embodiments, the pH of the medium is adjusted to a level ofapproximately from 3 to 8, including from about 5 to 6. In someembodiments, the culturing is carried out under aerobic conditions at aculturing temperature of generally from about 10 to 50° C., such as atabout 30° C., or about 37° . In some embodiments, the culturing iscarried out for a period of from 1 to 15 days, including from 3 to 7days. In general, any culturing method can be used that permits theproduction of CS-like compound, and the skilled practitioner will beable to select appropriate culture conditions depending on themicroorganisms to be cultured and the culturing method.

In some embodiments, cultivation comprises two stages, involvingdifferent media, such as a pre-culture media and main culture media, asillustrated in the examples below.

As noted above, suitable culture conditions can be selected by theskilled practitioner based on the microorganism being cultured. Forexample, as shown in Table 1 in Example 6 below, 10 ml of a pre-culturemedium can be added to a culture tube that is held at 30° C. for 1-2days with rotation of 200 rpm. Then the microorganism can be transferredto a suitable flask (such as a 100 ml or 300 ml flask) with 50 mlculture medium, and cultured at 30° C. for 3-5 days with rotation of 200rpm. These conditions are illustrative only.

Additional purification steps may optionally be performed to obtainisolated CS or CS-like compound, such as chromatography, including ionexchange chromatography, and/or filtration, including gel filtration,ultrafiltration, and alcohol precipitation, such as differential alcoholprecipitation or ethanol precipitation in the presence of salt.

Large scale fermentation procedures known in the art can be used toproduce commercial quantities of microbial-derived CS or CS-likecompound. Commercial batches may optionally be purified, such as byultrafiltration, and isolated CS or CS-like compound can be obtained,for example, by spray-drying the purified culture broth Alternatively,ethanol precipitation followed by vacuum drying will yield solidmicrobial-derived CS or CS-like compound.

Microbial strains that are particularly efficient at producing CS orCS-like compound can be developed by strain development procedures thatare known in the art, e.g., by multiple rounds of mutagenesis andselection of CS-secreting strains (using for example the DMMB assaydescribed below). In some embodiments, the mutagenesis does not involvegenetically engineering or recombinantly transforming strains to produceCS or CS-like compound.

Analysis and Characterization

CS content can be quantified and analyzed by any known method in theart. For example, CS can be detected and analyzed using various dyeassays, chromatographic tools, and electrophoretic techniques, includingthe chondroitinase treatment and HPLC and/or ELISA methodology describedbelow.

For example, numerous dye assays are known in the art that may be usedfor detecting and quantifying glycosaminoglycans. While in no waylimiting the present invention, suitable dye assays include thedimethylmethylene blue assay, the toluidine blue assay, and the Alcianblue assay. One suitable dimethylmethylene blue assay is illustrated inExample 1 below.

Various chromatography techniques may be used for quantifying andcharacterizing CS content and composition. For example, and in no waylimiting the invention, suitable chromatography techniques include highperformance liquid chromatography (HPLC), ion exchange chromatography,and size exclusion chromatography. ELISA methodologies also can be used,as illustrated in Example 8 below. In general, ELISA method can be usedto characterize samples that are less concentrated and not extensivelypurified. Suitable methodologies are illustrated in the examples below.

Electrophoretic techniques also may be used to analyze CS. For example,fluorophore-assisted carbohydrate electrophoresis (FACE) may be used toevaluate the degree and location of sulfation on the GAG chain.

As noted above and illustrated in Example 5 below, HPLC is used toanalyze CS from animal sources. Preliminary results indicate thatbacteria-derived CS or CS-like compound yields an HPLC pattern that issimilar to that of CS from animal sources. Alternatively, ELISAmethodologies can be used, as illustrated in Example 8.

As an alternative, CS can be hydrolyzed with acid and the resultingsugar components can be determined by HPLC. Because acid hydrolysis islikely to remove the sulfate groups, the sulfate content can bedetermined by determining the sulfur content of CS, using methods thatare known in the art.

Products

A microbial-derived CS or CS-like compound described herein can be usedin any compositions or methods where animal-derived CS is used, and inany compositions or methods where CS activity is desirable. For example,microbial-derived CS or CS-like compound can be formulated in apharmaceutical composition or nutraceutical composition furthercomprising, for example, pharmaceutically or nutraceutically acceptablecarriers and/or diluents. In some embodiments, the pharmaceutical ornutraceutical compositions are prepared in unit dosage forms comprisingany suitable dosage of the CS or CS-like compound. A microbial-derivedCS or CS-like compound also can be manufactured as a food additive andadded to any food product.

Methods

A microbial-derived CS or CS-like compound can be used in therapeutic orprophylactic methods, such as in methods for the treatment or preventionof osteoarthritis and/or for the maintenance of musculoskeletal health.Such methods may comprise administering a therapeutically effectiveamount of microbial-derived CS or CS-like compound to a patient in needthereof, wherein such a patient can be any mammal, including humans,suffering from or at risk of developing osteoarthritis, or desiring tomaintain musculoskeletal health using CS. As used herein,“therapeutically effective amount” means that amount that provides thespecific response for which the CS is administered. It is emphasizedthat a therapeutically effective amount will not always be effective intreating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art. Those skilled in the art can adjust such amounts inaccordance with standard practices as needed to treat a specific subjectand/or condition/disease.

The following examples are illustrative only, and in no respect limitthe invention. Other aspects of the invention will be apparent to thoseskilled in the art to which the invention pertains.

EXAMPLE 1 Dimethylmethylene Blue (DMMB) Assay for CS

Many versions of the DMMB assay exist. The DMMB assay described hereinhas good specificity for CS, and does not detect keratan sulfate,dermatin sulfate, hyaluronic acid or other polymers such as protein orDNA, due to the low pH and high salt concentration in the assay. Thus,the assay can be utilized to determine the amount of CS in crudesamples. See, e.g., Farndale et al., “Improved Quantization andDiscrimination of Sulphated Glycosaminoglycans by Use ofDimethylmethylene Blue,” Biochemica Et Biophysical Acta 883 173-77.(1986); Davies et al., “Characterization and Purification ofGlycosaminoglycans from Crude Biological Samples,” J. Agric. Food Chem.56: 343-48 (2008).

Materials: Dimethylmethylene blue (DMMB) hydrochloride can be purchasedfrom BioChemica International (Melbourne, Fla.). (An alternative sourceis 1,9-dimethylmethylene blue hydrochloride from Polysciences, Inc.(Warrington, Pa.)). Sodium chloride and glycine can be purchased fromSigma. Hydrochloric acid (w/w, 36.5-38%) can be purchased from FisherScientific. Chondroitin sulfate A sodium salt (from bovine trachea) canbe purchased from Sigma.

Preparation of DMMB: The dye can be prepared by dissolvingdimethylmethylene blue (4 mg) in a solution containing glycine (0.76 g),NaCl (0.5925 g), and 0.1 M HCl (23.75 mL) and deionized (DI) water(226.25 mL) to a total volume of 250 mL. The solution is stirred untilthe reagents are dissolved. The pH of the dye solution is pH 3.0 andabsorbance (A525) is about 0.300±0.06. The dye is stable for 3 monthswhile stored in a brown bottle at room temperature. A 0.1 M HCl solutionis prepared by taking stock HCl (w/w, 36.5-38%, ˜12M) (0.42 mL) anddissolving in deionized water (49.58 mL).

DMMB Procedure: Sample or Standard (40 μL) is added to a disposable 1 mLspectrophotometer cuvette containing dimethylmethylene blue dye solution(1 mL). The solution is mixed by inverting the parafilm covered cuvette5 times. The absorbance at 525 nm (A525) is read within the first fewseconds after stabilization, which happens within 10 seconds. Acalibration curve is prepared using chondroitin sulfate A sodium saltfrom bovine trachea (Sigma) as the standard. The assay has a linearrange of 0-4 μg/ml. The total time for the analysis of one sample shouldbe <1 minute from beginning to end to avoid inaccurate results due toprecipitation of the dye.

When assaying crude samples, samples can be assayed without dilution,unless the samples are known to contain high levels of CS. If theabsorbance reading is high (e.g., A₅₂₅˜0.7) dilutions can be used.

EXAMPLE 2 Isolation of Bacteria-Derived CS

Sterile nutrient broth (DIFCO), 100 ml, is inoculated with a smallportion (about 1 g) of natto obtained from a grocery store. The cultureis incubated at 37° C. with shaking (200 RPM) overnight (about 14hours). The culture is centrifuged at 3000 g for 30 minutes to pelletbacteria cells. The clear culture is removed and assayed for thepresence of CS-like compound by the DMMB assay described above. Theculture media is determined to contain 0.2 g CS/L (or CS-likecompound/L).

EXAMPLE 3 Determination of CS Size by Size Exclusion Chromatography

The size of CS is measured by size exclusion chromatography using aSuperdex 200 10/300GL column and an Akta Explorer FPLC chromatographysystem (GE Healthcare). Sample (0.5 ml) is injected on the column andthe column is run at 0.1 ml/min in 10 mM Tris HCl (pH 8.0) buffer.Protein is monitored at O.D. 280 nm and CS is monitored at O.D. 215 nm.Fractions are collected (1 ml) and assayed for the presence of CS by theDMMB assay. The molecular size is estimated by a pre-calibration of thecolumn with standard proteins of known size (GE Healthcare).

FIG. 1 shows the results of an analysis of the size distribution ofporcine CS by size exclusion chromatography, plotting CS content ascompared to fraction number. The molecular weight markers are anextrapolation from a calibration profile and linear regression linewhich relates elution volume (or fraction number) to molecular weight inkilodaltons (Kd).

FIG. 2 shows the results of an analysis of the size distribution ofbacteria-derived CS (obtained from the Naruse strain of Bacillus nattoas described in Example 7 below) by size exclusion chromatography,plotting CS content as compared to fraction number. The results indicatethat the bacteria-derived CS (obtained from the Naruse strain ofBacillus natto) has a molecular weight of about 10 Kd or less.

EXAMPLE 4 Characterization of CS by HPLC

Animal-derived CS can be characterized by a process that includeschondroitinase digestion and HPLC analysis of the resulting disaccharideunits. HPLC separates the disaccharide units of the polymer by sulfatecontent: disaccharides that contain no sulfate are eluted first,followed by disaccharides that contain sulfate in the 4 position ofN-acetyl-galactosamine (GalNac) and then disaccharides that containsulfate in the 6 position of GalNac. See, e.g., Sim et al.,“Quantitative analysis of chondrotin sulfate in raw materials,ophthalmic solutions, soft capsules and liquid preparations,” J.Chromatog. 818: 133-39 (2005).

For HPLC analysis, the CS polymer is first hydrolyzed (digested) bychondroitinase enzymes (available from Sigma or other vendors) intodisaccharide units. The resulting mixture is applied to a suitable HPLCcolumn, such as a Phenomenex Synergi 4u Polar-RP 80A column, and elutedwith a gradient of 1 mM tetrabutylammonium bisulfate to 50:50 1 mMtetrabutyammonium bisulfate: acetonitrile. The flow rate is 1 ml/min andO.D. is monitored at 240 nm. The column is calibrated with standardsulfated disaccharides (also available from Sigma).

FIG. 3 shows typical HPLC analysis of CS from bovine trachea (A), sharkcartilage (B), and porcine trachea (C), and from the Naruse strain ofBacillus natto (D). As the data show, the ratio of disaccharides withsulfates at the 4 position (4S) to disaccharides with sulfates at the 6position (6S) varies with the animal source of the CS. For example,shark CS has more 6S than 4S, while bovine and porcine CS have more 4Sthan 6S. Moreover, porcine CS has reproducibly higher amounts of 6S ascompared to bovine CS. The microbial-derived CS has similar 4S and 6Slevels. The 4S:6S ratio also may vary with the age of the animal orspecific tissue source of the CS, and, for microbial-derived CS, withthe culture conditions.

EXAMPLE 5 Ion Exchange Chromatography

Porcine or microbial-derived CS, such as bacteria-derived, is applied toa suitable ion exchange chromatography column, such as a DEAE FF 16/10column (GE Healthcare) and run on a Akta Explorer FPLC system (GEHealthcare). The flow rate is set at 1 ml/min and the column is elutedwith a linear buffer of 0-1 M NaCl in 10 mM Tris HCl (pH 8.0) buffer.The CS is monitored at O.D. 215 nm, and 2 ml fractions are collected andassayed for CS by the DMMB assay described above. The porcine CS bindsto the column tightly, eluting only at 1M salt. The bacteria-derived CSbinds to the column less tightly, eluting at about 0.5-0.7 M NaCl. Whilenot wanting to be bound by any theory, these results suggest thatbacteria-derived CS contains fewer sulfate groups than porcine CS.

EXAMPLE 6 Broth Library Screening of Strains Producing CS by DMMB Assay

Microbial strains that produce CS-like compound can be identified by thefollowing process, which is illustrative only. Candidate strains arecultured under conditions suitable for production of CS or CS-likecompound, as illustrated in Table 1 below, which depicts conditions fora two-stage production cultivation.

TABLE 1 Cultivation of Selected Strains Name Gentose pre-culture Mainculture Penicillium 8.3% B-ohgi medium Sun-grain medium multicolor 8.3%wheat bran (pH 6.5): Rhizopus oryzae (liquid culture) 1.5% LustergenFKRhizopus oryzae 10 ml/culture tube (soluble starch) Mucor javanicus 30°C. 200 rpm 2 days 0.3% MeastPIG Mucor (yeast extract) circinelloide f.circinelloide 0.3% KH₂PO₄ Mucor javanicus 0.1% MgSO₄•7H₂O Rhizomucor1.5% Sun-grain miehei (whiskey Rhizomucor fermentation residue miehei 50ml/300 mlΔflask Monascus 30° C. 200 rpm 5 days purpureus Saccharomycescerevisiae Mucor javanicus Penicillium 8.3% B-ohgi medium 5% wheat bran:funiculosum 10 ml/culture tube 5 g wheat bran/1.5 mL Penicillium 30° 200rpm 2 days 5% wheat roquefortii bran/100 mlΔflask Rhizomucor (SSF) 30°C. 5 days pusillus Monascus purpureus Aspergillus oryzae BB-1-84-38Penicillium camembertii Aspergillus − GPmedium (pH 5.7): DGL screeningniger 2.0% glucose medium Rhizopus oryzae − 0.5% Bacto peptone 50 ml/300mlΔflask Rhizopus oryzae − (DIFCO) 30° C. 200 rpm 5 days Mucor javanicus− 0.2% Yeast Extract Monascus − 0.05% MgSO₄•7H₂O purpureus 0.1% KH₂PO₄Monascus − 10 ml/culture tube purpureus 30° C. 200 rpm 2 daysSchizosaccharo − myces pombe Candida − tropicalis Candida − tropicalisSaccharomyces − cerevisiae Candida − albicans Saccharomyces − cerevisiaeSaccharomyces − cerevisiae Aspergillus + oryzae Aspergillus + nigerAspergillus + awamori Penicillium + multicolor Rhizopus oryzae +Rhizomucor + pusillus Rhizomucor + miehei Rhizomucor + mieheiEscherichia coli − Tryptic soy broth (DIFCO) Bacteria screening Bacillus− (pH 7.3 ± 0.2 (25° C.)): medium (pH 7): licheniformis 3% Tryptic soybroth 1% Polypeptone Escherichia coli − (DIFCO) 0.25% yeast extractEscherichia coli − 10 ml/culture tube (DIFCO) Bacillus − 30° C. 200 rpm1 day 0.10% (NH₄)₂SO₄ licheniformis 0.05% KH₂PO₄ 501SMN11- 0.025%MgSO₄•7H₂O MCI12 0.0001% CaCl₂•2H₂O Bacillus − (optionally, 0.5%circulans gentiooligosaccharide) Bacillus subtilis + 50 ml/300 mlΔflaskEscherichia coli + 30° C. 200 rpm 3 days Bacillus + licheniformisEscherichia coli + Escherichia coli + Bacillus + circulans

Sample broths from different genus and different species can be assayedfor CS-like compound production. As outlined in FIG. 4, a first assayscreened 2,208 sample broths from 34 different genus and 240 differentspecies using the DMMB assay as described in Example 1. From thisscreen, 400 sample broths tested positive for CS-like compoundproduction. From these 400 sample broths, sample broths from 10different genus (Aspergillus, Bacillus, Candida, Eschericia, Monascus,Mucor, Penicillium, Rhizomucor, Rizopus, and Saccharomyces) wereselected for further analysis. As shown in Table 2 below, 26 sampleswere analyzed for 4S:6S ratio by ELISA (as described in Example 8 below)and 19 of these samples were analyzed for CS-like compound molecularweight determination.

TABLE 2 First Screening Results for CS-Producing Microbial Strains Sam-Fermentation DMMB MW ple. Method Strain Name Assay 4S/6S (daltons) 1Fungi (solid state Monascus 0.110 0.37 fermentation) purpureus 2 Fungi(solid state Aspergillus 0.129 0.1 33,000-5400 fermentation) oryzaeBB-1-84-38 3 Eucarya Mucor 0.139 6.02 10,000-1000 (submerged javanicusfermentation) 4 Fungi (solid state Penicillium 0.134 0.08 18,000-5400fermentation) camembertii 5 Eucarya Mucor 0.137 3.08 (submergedcircinelloides fermentation) f. circinelloides 6 Eucarya Mucor 0.1440.44  3000-360 (submerged javanicus fermentation) 7 Eucarya Rhizomucor0.073 0.08  1400-360 (submerged miehei fermentation) 8 EucaryaRhizomucor 0.071 0.06 10,000-1400 (submerged miehei fermentation) 9Fungi (solid state Penicillium 0.219 0.03 10,000-1200 fermentation)roquefortii 10 Eucarya Monascus 0.031 6.19  1400-360 (submergedpurpureus fermentation) 11 Fungi (solid state Rhizomucor 0.071 0.3913,400-2200 fermentation) pusillus 12 Fungi (solid state Penicillium0.229 0.12 13,400-4000 fermentation) funiculosum 13 Eucarya Mucor 0.1470.34 19,000-1400 (submerged javanicus fermentation) 14 Eucarya Rhizopus0.027 3.44 18,000-3000 (submerged oryzae fermentation) 15 EucaryaRhizopus 0.056 0.15 24,500-4000 (submerged oryzae fermentation) 16Eucarya Rhizopus 0.036 0.059 24,500-4000 (submerged oryzae fermentation)17 Eucarya Mucor 0.064 0.22 18,000-4000 (submerged javanicusfermentation) 18 Eucarya Rhizomucor 0.079 0.15 13,400-1000 (submergedpusillus fermentation) 19 Eucarya Rhizomucor 0.039 5.52 13,400-900 (submerged miehei fermentation) 20 Eucarya Monascus 0.017 6.7910,000-1200 (submerged purpureus fermentation) 21 Bacteria Bacillus0.144 0.24 (submerged subtilis fermentation) 22 Bacteria Escherichia0.193 (submerged coli fermentation) 23 Bacteria Escherichia 0.180 0.66(submerged coli fermentation) 24 Bacteria Bacillus 0.245 0.018(submerged licheniformis fermentation) 25 Bacteria Bacillus 0.20518,000-5400 (submerged licheniformis fermentation) 501SMN11- MCI1 26Bacteria Bacilluks 0.150 2.92 (submerged circulans fermentation)

Based on the screening described above and a second screening, 132microbial strains were selected as CS (or CS-like compound) productionstrains. Tables 3-4 set forth these strains and the amount of CS (orCS-like compound) detected in their culture media (ng/mL) as derivedfrom a standard curve using the DMMB assay. Table 3 lists bacterialstrains and Table 4 lists fungal strains.

TABLE 3 CS-Producing Bacteria CS Bacterial Species (ng/ml) Bacilluscereus 8.6 Bacillus circulans 11.7 Bacillus coagulans 8.5 Bacilluslicheniformis 9.0 Bacillus megaterium 8.8 Bacillus subtilis 8.9Lactobacillus acidophilus 8.9 Corynebacterium glutamicum 80.8Microbacterium arborescens 8.0 Micrococcus varians 8.7 Monascuspurpureus 10.1 Streptomyces 8.3 olivochromogenes Streptomyces rubginosus8.0 Escherichia coli 8.8

TABLE 4 CS-Producing Fungi CS Fungal Species (ng/ml) Aspergillus repens8.17 Aspergillus sydowi 8.72 Aspergillus aculeatus 92.36 Aspergillusjaponicus 92.68 Aspergillus kawachii 82.68 Aspergillus melleus 93.64Aspergillus nidulans 8.18 Aspergillus niger var. Intermedius 80.28Aspergillus phoenicis 93.88 Aspergillus pulverulentus 91.88 Aspergillussulphureus var. minimus 86.68 Aspergillus terreus 8.18 Aspergillusterricola 90.20 Aspergillus usamii 88.84 Aspergillus versicolor 93.08Aspergillus wentii 95.00 Aspergillus aureus 95.08 Aspergillus awamori9.66 Aspergillus candidus 95.40 Aspergillus chevalieri 80.36 Aspergillusfavipes 8.32 Aspergillus fisheri 8.98 Aspergillus flavus 125.96Aspergillus nakazawai 95.16 Aspergillus niger 11.14 Aspergillus oryzae11.10 Aspergillus phoenicis 84.52 Aspergillus sojae 8.02 Aspergillus spBB46-2-22 9.21 Candida albicans 11.84 Candida cylindracea 9.99 Candidafermentati 11.65 Candida guilliermondii 84.88 Candida intermedia 8.10Candida kefyr 8.83 Candida krusei 8.74 Candida maltosa 8.34 Candidamycoderma 8.64 Candida pelliculosa 8.94 Candida rugosa 10.64 Candidatenuis 82.08 Candida tropicalis 11.50 Candida utilis 8.96 Cryptococcusalbidus var. Diffluens 8.35 Cryptococcus laurentii 8.74 Debariomyceshansenii 10.11 Debaryomyces kloekeri 9.41 Debaryomyces polymorphus 82.24Dipodascus magnusii 8.65 Endomycopsella vini 81.52 Endomycopsiscapsularis 8.34 Eurotium chevalieri 82.12 Galactomyces reessii 8.87Geotrichum candidum 81.88 Hansenula carifornica 8.66 Hasegawaea japonica8.64 Kluveromyces marxianus 8.50 Kluyveromyces fragilis 8.15Kluyveromyces lactis 11.56 Kluyveromyces marxianus 8.36 Mortierellavinacea 8.49 Mucor circinelloides f. circinelloides 9.10 Mucor javanicus10.73 Penicillium camembertii 9.14 Penicillium funiculosum 8.68Penicillium lilacinum 86.44 Penicillium multicolor 8.99 Penicilliumroquefortii 8.69 Pichia anomala 9.98 Pichia farinosa 10.00 Pichiamembranaefaciens 9.42 Rhizomucor miehei 8.86 Rhizomucor pusillus 8.48Rhizopus acetoinus 82.52 Rhizopus achlamydosporus 94.20 Rhizopuschinensis 88.44 Rhizopus chungkuoensis 84.12 Rhizopus chuniang 86.76Rhizopus delemar 88.84 Rhizopus formosaensis 88.52 Rhizopus hangchow89.08 Rhizopus jaranicus 90.28 Rhizopus kansho 88.20 Rhizopusmicrosporus 85.80 Rhizopus nigricans 84.92 Rhizopus niveus 8.10 Rhizopusnodosus 89.08 Rhizopus oligosporus 8.05 Rhizopus oryzae 8.78 Rhizopuspeka II 87.48 Rhizopus peka I 89.64 Rhizopus pseudochinensis 89.56Rhizopus reflexus 85.96 Rhizopus salebrosus 87.96 Rhizopus shanghaiensis83.08 Rhizopus stolonifer 8.05 Rhizopus tamarii 86.84 Rhizopus thermosus86.68 Rhizopus tonkinensis 83.56 Rhodotorula glutinis 8.35 Rhodotorulamucilaginosa 8.57 Saccharomyces cerevisiae 11.50 Saccharomycesdelbrueckii var. 9.36 Mongolicus Saccharomyces fragilis 8.42Saccharomyces lactis 8.30 Saccharomyces marxianus 8.52Schizosaccharomyces pombe 9.41 Streptomyces fradiae No. 15 8.02Talaromyces emersonii 8.21 Torulopsis dattila 9.90 Torulopsis globosa8.94 Trichoderma reesei 8.28 Trichoderma viride 8.04 Trichosporoncutaneum 10.68 Yamadazyma farinosa 8.16 Yamadazyma guilliermondii 9.31Yarrowia lipolytica 83.60 Zygosaccharomyces rouxii 8.54

EXAMPLE 7 CS-like Compound Production By Various Bacillus natto Strains

To assess production of CS-like compound across various Bacillus nattostrains, five different strains were analyzed for microbial CS-likecompound production using the methods described in Example 2. MicrobialCS-like compound production was found for three strains: the Miyagino,Naruse, and Takahashi strain. Two strains, Daiwa strain 1 and Daiwastrain 2, did not produce a CS-like compound.

Purified Naruse CS-like compound was analyzed by HPLC as described inExample 4 and the results are shown in FIG. 3(D). Notably, the 4S peak(first peak) and the 6S peak (second peak) are of similar intensity,indicating similar amounts of 4S and 6S, which is different from animalCS (porcine or bovine or shark).

EXAMPLE 8 ELISA Analysis of CS Digestion Products

CS or CS-like compound derived from bovine, Bacillus natto Narusestrain, and porcine (Zeria Pharmaceutical Co., Ltd. Japan) were digestedwith chondroitinase AC (EC 4.2.2.5) (Sigma) as described in Example 4,except the digestion products were not analyzed by HPLC. Instead, thedigestion products were immobilized in a microtiter well by standardELISA procedures and analyzed by antibodies to 4S and 6S (Millipore).The antibodies are mouse monoclonal antibodies which can be detected byanti-mouse horseradish peroxidase conjugated antibody (Sigma) anddetected by fluorescence using the QuantaBlu detection kit (ThermoFisher).

As shown in FIG. 5, the 4S/6S pattern for each CS is similar to thatobserved by HPLC analysis. The advantage of the ELISA method is that itcan be used to characterize samples that are less concentrated and notextensively purified.

EXAMPLE 9 CS-like Compound Production By Various Bacillus Strains

The production of CS-like compound across various Bacillus strains wasassessed as described in Example 2. Microbial CS-like compoundproduction was found for 73 strains, as illustrated in Table 5 below,which also shows the amount of CS (or CS-like compound) detected intheir culture media (μg/mL) as derived from a standard curve using theDMMB assay.

TABLE 5 CS-Producing Bacillus Strains CS Bacillus Strain (μg/mL)Bacillus alvei 62.0 Bacillus amyloquefaciens 419.8 Bacillusaneurinolyticus 62.0 Bacillus atrophaeus 9.0 Bacillus brevis 86.1Bacillus cereus BA-1 22.0 Bacillus cereus K681 103.4 Bacillus cereusvar. mycoides 25.0 Bacillus circulans 66.4 Bacillus coagulans 14.2Bacillus firmus 103.4 Bacillus flexus 74.0 Bacillus flexus BA2 77.0Bacillus fusiformis 27.0 Bacillus halodurans 67.0 Bacillus licheniformis94.1 Bacillus licheniformis CE207 13.0 Bacillus licheniformis CE262201.0 Bacillus megaterium GDH-1 46.0 Bacillus megaterium No. 3344 52.0Bacillus mycoides 30.0 Bacillus polymyxa 1.0 Bacillus polymyxa EH-4 3.0Bacillus pumilus 117.0 Bacillus racemilacticus 89.0 Bacillus roseus 78.1Bacillus sp. 100.0 Bacillus sp. A-15 74.0 Bacillus sp. A-3 71.0 Bacillussp. CE-207-1 12.0 Bacillus sp. CE-207-4 15.0 Bacillus sp. CE-262-7 250.0Bacillus sp. CE-262-C 244.0 Bacillus sp. L1HT1 73.0 Bacillus sp. No. 12818.0 Bacillus sp. No. 56Y001DA011-4 27.0 Bacillus sp. No. 71Y001DA023-019.0 Bacillus sp. Origin 34 80.0 Bacillus sp. Origin 6 85.0 Bacillussphaericus 90.0 Bacillus sphaericus 19.0 Bacillus stearothermophilus30.4 Bacillus subtilis 103.0 Bacillus subtilis (natto sawamura) 89.6Bacillus subtilis (natto) 237.7 Bacillus subtilis A-1 83.0 Bacillussubtilis A-10 79.0 Bacillus subtilis A-11 73.0 Bacillus subtilis A-1297.0 Bacillus subtilis A-13 77.0 Bacillus subtilis A-14 111.0 Bacillussubtilis A-2 114.0 Bacillus subtilis A-3 95.0 Bacillus subtilis A-4 80.0Bacillus subtilis A-5 104.0 Bacillus subtilis A-6 103.0 Bacillussubtilis A-7 143.0 Bacillus subtilis A-8 82.0 Bacillus subtilis A-9 91.0Bacillus subtilis K wild 86.1 Bacillus subtilis Marburg W23 SM 215.9Bacillus subtilis naruse 171.3 Bacillus subtilis R1 109.0 Bacillussubtilis R2 128.0 Bacillus subtilis S3 164.0 Bacillus subtilis subsp.subtilis 94.0 Bacillus subtilis var. atterimus 799.5 Bacillus subtilisvar. niger 137.8 Bacillus thermoamyloliquefaciens 11.5 Bacillusthermodenitrificans 12.2 Bacillus thermoproteolyticus BT-9 31.8 Bacillusthuringiensis 22.0 Bacillus natto 100.0

1. A method for producing a chondroitin sulfate-like compound, comprising (a) culturing a microbial culture under conditions suitable for chondroitin sulfate production; and (b) obtaining chondroitin sulfate from said culture.
 2. The method of claim 1, wherein said microbial culture comprises a bacterium or a fungus.
 3. The method of claim 1, wherein said microbial culture comprises a naturally occurring bacterium or a naturally occurring fungus.
 4. The method of claim 3, wherein said bacterium is from a genus selected from the group consisting of Corynebacterium, Microbacterium, Micrococcus, Monascus, Streptomycetes, Escherichia, Bacillus and Lactobacillus.
 5. The method of claim 4, wherein said bacterium is from the genus Bacillus.
 6. The method of claim 5, wherein said bacterium is from a strain of Bacillus natto.
 7. The method of claim 6, wherein said strain of Bacillus natto is selected from the group consisting of the Naruse strain, the Miura strain and the Takahashi strain.
 8. The method of claim 3, wherein said fungus is from a genus selected from the group consisting of Aspergillus, Endomycopsella, Endomycopsis, Hansenula, Hasegawaea, Penicillium, Pichia, Monascus, Candida, Debaryomyces, Eurotium, Galactomycetes, Geotrichum, Rhodotorula, Saccharomyces, Trichoderma, Kluveromyces, Schizosaccharomyces, Streptomyces, Talaromyces. Torulopsis, Yamadazyma, Yarrowia, Zygosaccharomyces, Mucor, Mortierella, Rhizomucor, Rhizopus, Cryptococcus, Dipodascus, and Trichosporon.
 9. The method of claim 1, wherein said bacterial culture comprises natto.
 10. The method of claim 1, wherein said culturing comprises incubating said microbial culture in nutrient broth at 37° C.
 11. The method of claim 1, wherein said culturing comprises incubating said microbial culture in culture medium at 30° C.
 12. The method of claim 1, wherein said culturing comprises two stages, comprising a first stage comprising culturing in a pre-culture medium and a second stage comprising culturing in a main culture medium.
 13. The method of claim 1, wherein said obtaining comprises a method selected from the group consisting of centrifugation to remove microbial cells, filtration, chromatography, and alcohol precipitation.
 14. An isolated chondroitin sulfate-like compound produced by the method of claim
 1. 15. The isolated chondroitin sulfate-like compound of claim 14, wherein the compound is chondroitin sulfate.
 16. The isolated chondroitin sulfate-like compound of claim 14, wherein said chondroitin sulfate-like compound comprises N-acetyl-galactosamine groups having a sulfate group at the 4 position and N-acetyl-galactosamine groups having a sulfate group at the 6 position.
 17. A composition comprising an isolated chondroitin sulfate-like compound produced by the method of claim
 1. 18. The composition of claim 17, wherein said composition is a pharmaceutical composition, nutraceutical composition, or food product.
 19. An isolated microbial-derived chondroitin sulfate-like compound.
 20. The microbial-derived chondroitin sulfate-like compound of claim 19, wherein said chondroitin sulfate-like compound comprises N-acetyl-galactosamine groups having a sulfate group at the 4 position and N-acetyl-galactosamine groups having a sulfate group at the 6 position, wherein the ratio of disaccharides with sulfate at the 4 position (4S) to disaccharides with sulfate at the 6 position (6S) is greater than
 1. 21. The microbial-derived chondroitin sulfate-like compound of claim 19, wherein said chondroitin sulfate-like compound comprises N-acetyl-galactosamine groups having a sulfate group at the 4 position and N-acetyl-galactosamine groups having a sulfate group at the 6 position, wherein the ratio of disaccharides with sulfate at the 4 position (4S) to disaccharides with sulfate at the 6 position (6S) is less than
 1. 22. The microbial-derived chondroitin sulfate-like compound of claim 19, wherein said chondroitin sulfate-like compound has a molecular weight selected from (i) about 300 to about 3,000 daltons; (ii) about 1,000 to about 10,000 daltons; (iii) about 500 to about 15,000 daltons; and (iv) about 1,000 to about 20,000 daltons; (iv) about 1,000 to about 25,000 daltons; (iv) about 5,000 to about 35,000 daltons.
 23. The microbial-derived chondroitin sulfate-like compound of claim 19, wherein said compound is chondroitin sulfate.
 24. A method for the treatment or prevention of osteoarthritis, or for the maintenance of musculoskeletal health, comprising administering to a patient in need thereof a therapeutically effective amount of a chondroitin sulfate-like compound produced by the method of claim
 1. 